Our goal is to elucidate the mechanisms of action of OX40 (CD134), a new costimulatory molecule that belongs to the TNF-R superfamily, in T cell activation and T cell regulation, and the therapeutic implication of targeting 0X40 in the induction of transplant tolerance. This goal is based on our recent discovery that OX40 regulates a critical but poorly understood process in transplant rejection. It is known that OX40 is expressed on activated but not resting T cells, and 0X40 signals support cell survival and proliferation. However, we found that memory T cells that are alloreactive and the CD4+Foxp3+ Tregs that are required for tolerance induction constitutively express OX40 at high levels. Furthermore, we have new data showing that OX40 defines a population of Foxp3+ Tregs and T effectors, and these OX40 cells can either mediate rejection or tolerance in an adoptive transfer model depending on whether the OX40 receptor is intentionally blocked or deliberately stimulated. Our new data also showed that OX40 costimulation can profoundly alter the regulatory functions of naturally arising CD4+CD25+Tregs and can prevent the induction of new Foxp3 Tregs from activated effector T cells. Based on these findings, we generated the following hypothesis: OX40 controls a critical checkpoint in the development of a regulatory type of immune response in transplant models. The profound effects of OX40 in favoring a strong effector and memory types of immunity may block the regulatory functions of CD4+CD25+Foxp3+Tregs or prevent the induction of new inducible Foxp3+ Tregs from the effector pool. This hypothesis will be tested via the following 4 Aims. Aim# 1. To test the hypothesis that OX40 reprograms the natural FoxpS* Tregs to a non-regulatory and inflammatory phenotype or OX40 is pro-apoptotic to CD4*Foxp3+ natural Tregs by directly modifying their life and death programs. Aim # 2. To test the hypothesis that OX40 prevents de novo generation of new inducible Foxp3+ Tregs by promoting differentiation of effector T cells to Th1/Th2 effector T cells or to memory type of T cells. Aim #3. To test the hypothesis that fully differentiated effector/memory T cells instructed by OX40 costimulation are highly resistant to Foxp3+Treg mediated suppression. Aim # 4. To test the hypothesis that blocking the OX40/OX40L pathway readily induces dominant transplant tolerance by favoring a regulatory type of immune response It is anticipated that accomplishment of these studies will provide critical insights in the development of new therapeutic strategies aimed at manipulating Tregs in the creation of transplant tolerance and in the treatment of certain autoimmune diseases and cancers.